Calcium-dependent regulation of the motor activity of recombinant full-length Physarum myosin.

We successfully synthesized full-length and the mutant Physarum myosin and heavy meromyosin (HMM) constructs associated with Physarum regulatory light chain and essential light chain (PhELC) using Physarum myosin heavy chain in Sf-9 cells, and examined their Ca(2+)-mediated regulation. Ca(2+) inhibited the motility and ATPase activities of Physarum myosin and HMM. The Ca(2+) effect is also reversible at the in vitro motility of Physarum myosin. We demonstrated that full-length myosin increases the Ca(2+) inhibition more effectively than HMM. Furthermore, Ca(2+) did not affect the motility and ATPase activities of the mutant Physarum myosin with PhELC that lost Ca(2+)-binding ability. Therefore, we conclude that PhELC plays a critical role in Ca(2+)-dependent regulation of Physarum myosin.

Calcium regulation of the ATPase activity of Physarum and scallop myosins using hybrid smooth muscle myosin: the role of the essential light chain.

To examine the role of two light chains (LCs) of the myosin II on Ca2+ regulation, we produced hybrid heavy meromyosin (HMM) having LCs from Physarum and/or scallop myosin using the smooth muscle myosin heavy chain. Ca2+ inhibited motility and ATPase activity of hybrid HMMs with LCs from Physarum myosin but activated those of hybrid HMM with LCs from scallop myosin, indicating an active role of LCs. ATPase activity of hybrid HMMs with LCs from different species showed the same effect by Ca2+ even though they did not support motility. Our results suggest that communication between the original combinations of LC is important for the motor function.

[Involvement of extracellular cAMP-specific phosphodiesterase in control of motile activity of Physarum polycephalum plasmodium].

Possible involvement of extracellular cAMP-specific phosphodiesterase in the control of cell motile behavior has been investigated in Physarum polycephalum plasmodium, a multinuclear amoeboid cell with the autooscillatory mode of motility. It was found that the rate of the hydrolysis of 10 mM cAMP by a partially purified preparation of cAMP-specific phosphodiesterase secreted by the plasmodium in the course of migration decreases 20-30 times under the action of 1 mM dithiothreitol. In the presence of 1-5 mM of this strong reducing agent, the onset of the plasmodium spreading and the transition to the stage of migration were delayed in a concentration-dependent manner. In accordance with the morphological pattern of motile behavior, the duration of the maintenance of high frequency autooscillations, which normally precede the increase in the rate of the spreading and appear also in response to the application of attractants at spatially uniform concentrations, strongly increased by the action of dithiothreitol. The results obtained suggest that the autocrine production of cAMP and extracellular cAMP-specific phosphodiesterase is an important constituent of the mechanism controlling the motile behavior of the Physarum polycephalum plasmodium.

Interaction between cell shape and contraction pattern in the Physarum plasmodium.

The relationship between cell shape and rhythmic contractile activity in the large amoeboid organism Physarum polycephalum was studied. The organism develops intricate networks of veins in which protoplasmic sol moved to and fro very regularly. When migrating on plain agar, the plasmodium extends like a sheet and develops dendritic veins toward the rear. After a particular stimulation, the vein organization changes into veinless or vein-network structures. In both structures, the mixing rate of the protoplasm, which is related to communication among contraction oscillators, decreased compared with that of the dendritic one. Accompanying these changes in vein structure, the spatio-temporal pattern of the rhythmic contraction changed into a small-structured pattern from a synchronized one. In the above process, cell shape affects the contraction pattern, but, conversely, the contraction pattern effects the cell shape. To demonstrate this, a phase difference in the rhythmic contraction was induced artificially by entraining the intrinsic rhythm to external temperature oscillations. New veins then formed along the direction parallel to the phase difference of the rhythm. Consequently, the vein organization of the cell interacts with the contractile activity to form a feedback loop in a mechanism of contraction pattern formation.

Relationship between intracellular period modulation and external environment change in Physarum plasmodium.

The relationship between intracellular period modulation and external environment change was investigated from the viewpoint of internal information coding in Physarum plasmodium. For the external conditions, concentration changes of attractant (galactose) and repellent (KCl) were used, and the internal responses were measured as the thickness oscillation of the plasmodium. (i) Period of the intracellular oscillation decreased when the concentration of attractant was increased and when the concentration of repellent was decreased. (ii) The period increased when the attractant was decreased and when the repellent was increased. (iii) The larger concentration change induced the larger period modulation. (iv) These responses were observed when the change of concentration was greater than a threshold value. From these results, it was clarified that the relative change in environmental condition is encoded on the relative period modulation in intracellular oscillation. This means that the period change does not directly represent the environment itself but represents the change of its condition. Thus, it is further suggested that the plasmodium estimates the environmental condition based on the relationship between the previous external condition and the present one.

Entrainment to external Ca2+ oscillation in ionophore-treated Physarum plasmodium.

To elucidate the mechanism of mutual interaction between intracellular chemical rhythms in the Physarum plasmodium, external Ca2+ oscillation was applied to the ionophore-treated plasmodial strand and its response was measured as tension oscillation. (i) Tension oscillation is entrained and phase locked to the externally applied Ca2+ oscillation. (ii) Two kinds of stable phase relationship, in-phasic and anti-phasic ones, are observed between them. (iii) Transition between the two stable phase relationships is also observed. These results suggest that intracellular rhythms with control tension generation are mutually entrained by means of cytosolic Ca2+ oscillation in the organism and that their interactions have two kinds of stable phase relationships.

Purification of a novel Ca-binding protein that inhibits myosin light chain kinase activity in lower eukaryote Physarum polycephalum.

Myosin light chain kinase (MLCK) was partially purified from the lower eukaryote Physarum polycephalum. The activity to phosphorylate Physarum myosin was maximal in the absence of Ca2+ and decreased with an increase in Ca2+ concentration with a microM-level Kd. The Ca-binding protein contained in the MLCK preparation was purified to homogeneity. The native protein had a molecular mass of 75 kDa, while under denaturing conditions, it was 38 kDa. Ca-dependent changes in the intensities of intrinsic fluorescence showed that the Kd of the protein for Ca2+ was also in the microM-range. Our results suggest that the Ca-binding protein would play a key role in the effects of Ca2+ in the MLCK preparation.

Acid-soluble purine and pyrimidine derivatives of growing, encysting and encystment-inhibited amoebae.

The intracellular acid-soluble purine and pyrimidine derivatives of myxamoebae-swarm cells of Physarum flavicomum were investigated during growth, microcyst formation, and during adenine-inhibition of encystment, using high performance liquid chromatography (HPLC). We also studied the incorporation of exogenous radioactive adenine into the acid soluble purine derivatives and S-adenosyl-sulphur compounds separated by HPLC. The most abundant ribonucleoside monophosphate was AMP in the growing and 15 h encysting cells (NC), while it was UMP in the 15 h adenine-inhibited cells (AIC). ADP was the nucleoside diphosphate present in the greatest quantity in the growing and NC cells but it was CDP in the AIC. The nucleoside triphosphate in highest concentration was ATP, UTP, and GTP in growing, NC, and AIC, respectively. Guanosine was the most abundant nucleoside in all cells. The nucleobase occurring in greatest concentration was cytosine, cytosine and guanine, and adenine in the growing, NC, and AIC, respectively. The AMP content in the 15 h AIC was 2.1-fold higher than that of adenosine. The 15 h NC had the lowest adenylate energy charge, a value of 0.54 +/- 0.02, while the values for growing cells and the AIC were 0.62 +/- 0.02 and 0.76 +/- 0.01, respectively. [14C]-Adenine labelling studies (15 h) revealed the occurrence of purine nucleotide interconversion, as the label was detected not only in adenosine, AMP, ADP, ATP, but also in guanine, guanosine, GMP, GDP, GTP, as well as, in inosine monophosphate and xanthosine monophosphate. The percentage incorporation of the radiolabelled adenine into AMP was higher than into adenosine. An increased intracellular level of guanine nucleotides is associated with the inhibition of encystment. The extracellular adenine, rather than internal adenine sources, appears to be the primary precursor of nucleotide for S-adenosylmethionine synthesis during adenine-inhibition of encystment.

Intracellular polyamine patterns during encystment of Physarum flavicomum.

In Physarum flavicomum Berk., growing amoebae convert to dormant cysts under conditions of nutrient imbalance. Exogenous adenine inhibits the process and the cells produce an elevated intracellular concentration of S-adenosylmethionine. Evidence indicates that the increased level of S-adenosylmethionine is responsible for the disruption of the normal developmental process. One of the biological functions of S-adenosylmethionine is in polyamine synthesis and it is known that a well-controlled intracellular concentration of polyamines is essential for normal cell growth and differentiation. In this study, high-performance liquid chromatography was used to determine the intracellular polyamine patterns in growing cells, adenine-treated and normal encysting cells, and dormant cysts. Putrescine and spermidine were the most abundant polyamines found in the cells; growing cells had the highest level, adenine-treated cells had a 1.5 to 2.0 times higher level than normal encysting cells, while cysts had the lowest (only 3 and 12% of that of growing cells). Cadaverine and N1-acetylspermidine were found in all the cells and their levels decreased during encystment. Acetylputrescine was found in growing cells only and acetylspermine was found in all cells except cysts. Acetylcadaverine, N8-acetylspermidine, 1,3-diaminopropane, and spermine were not detected in any of the cells.

Changes in phospholipid composition and phospholipase D activity during the differentiation of Physarum polycephalum.

Changes in phospholipid composition and phospholipase D activity were observed during a differentiation from haploid myxoamoebae to diploid plasmodia of a true slime mold, Physarum polycephalum. In the amoeboid stage, the main components of phospholipid fraction were phosphatidylethanolamine (PE, 43.3%), phosphatidylcholine (PC, 28.8%) and phosphatidylinositol (PI, 8.0%), but in the plasmodial stage, PC was dominant (40.7%) and other main components were PE (31.5%) and phosphatidic acid (PA, 11.0%). The specific activity of phospholipase D in the plasmodia was 5.7-times higher than that in the myxoamoebae when measured in the presence of Ca2+ at the alkaline pH. In the amoeboid stage, phospholipase A activity (A1 or A2) was detected at the alkaline pH with Ca2+. Phospholipase D activity in the plasmodia was characterized: pH optimum was 6.0; Ca2+ was required for the reaction and Ba2+ could substitute partly for Ca2+; PE was the best substrate for the hydrolytic activity and PC and PI were not appreciably hydrolyzed; and all detergents tested inhibited the enzyme activity.

S-adenosylmethionine and S-adenosylhomocysteine transitions in encysting Physarum flavicomum amoebae.

Previous studies from this laboratory suggested that in Physarum flavicomum an S-adenosylmethionine (SAM) related metabolic flux is involved in the developmental control of microcyst formation. In the present study, this phenomenon was further analyzed by comparing the metabolic effects of compounds found to favor, stimulate, or inhibit the normal encystment process. The compounds utilized were adenine, cycloleucine, L-ethionine, DL-selenomethionine, cadaverine, cyclohexylammonium sulfate, papaverine, mycophenolic acid, decoyinine, putrescine, spermidine, and spermine. Following incubation with the compounds or combinations of the compounds, the intracellular levels of SAM and SAM metabolites were determined using high performance liquid chromatography. The actual level of the intracellular SAM pool varied considerably (notably after 15 h of incubation), depending on the compound(s) in the incubation solution, but was always highest during inhibition of encystment. SAM was undetectable or barely detectable during stimulation of encystment or under conditions favoring encystment. In cells exposed to L-ethionine, we detected intracellular S-adenosylethionine rather than SAM. The intracellular methionine pool in cells encysting normally for 15 h was found to be 1.3 mumol/pg DNA. Both SAM and S-adenosylhomocysteine (SAH) were present in growing amoebae at concentrations greater than that of cells undergoing normal encystment. The developmental transition from growing amoebae to dormant cysts in P. flavicomum is metabolically characterized by the adjustment of the intracellular concentration of SAM and SAH to a minimal critical level of 0.31 and 0.70 fmol/pg DNA, respectively.

An unusual polyanion from Physarum polycephalum that inhibits homologous DNA polymerase alpha in vitro.

From extracts of microplasmodia of Physarum polycephalum and their culture medium, an unusual substance was isolated which inhibited homologous DNA polymerase alpha of this slime mold but not beta-like DNA polymerase and not heterologous DNA polymerases. Analysis, especially NMR spectroscopy, revealed the major component to be an anionic polyester of L-malic acid and the inhibition to be due to poly(L-malate) in binding reversibly to DNA polymerase alpha. The mode of inhibition is competitive with substrate DNA and follows an inhibition constant Ki = 10 ng/mL. Inhibition is reversed in the presence of spermine, spermidine, poly(ethylene imine), and calf thymus histone H1. According to its ester nature, the inhibitor is slightly labile at neutral and instable at acid and alkaline conditions. Its largest size corresponds to a molecular mass of 40-50 kDa, but the bulk of the material after purification has lower molecular masses. The inhibitory activity depends on the polymer size and has a minimal size requirement.

Evidence for active interactions between microfilaments and microtubules in myxomycete flagellates.

We have previously observed the apparent displacement of microfilaments over microtubules in the backbone structure of permeabilized flagellates of Physarum polycephalum upon addition of ATP (Uyeda, T. Q. P., and M. Furuya. 1987. Protoplasma. 140:190-192). We now report that disrupting the microtubular cytoskeleton by treatment with 0.2 mM Ca2+ for 3-30 s inhibits the movement of the microfilaments induced by subsequent treatment with 1 mM Mg-ATP and 10 mM EGTA. Stabilization of microtubules by pretreatment with 50 microM taxol retarded both the disintegrative effect of Ca2+ on the microtubules and the inhibitory effect of Ca2+ on the subsequent, ATP-induced movement of the microfilaments. These results suggest that the movement of the microfilaments depends on the integrity of the microtubular cytoskeleton. EM observation showed that the backbone structure in control permeabilized flagellates consists of two arrays of microtubules closely aligned with bundles of microfilaments of uniform polarity. The microtubular arrays after ATP treatment were no longer associated with microfilaments, yet their alignment was not affected by the ATP treatment. These results imply that the ATP treatment induces reciprocal sliding between the microfilaments and the microtubules, rather than between the microfilaments themselves or between the microtubules themselves. While sliding was best stimulated by ATP, the movement was partially induced by GTP or ATP gamma S, but not by ADP or adenylyl-imidodiphosphate (AMP-PNP). AMP-PNP added in excess to ATP, 50 microM vanadate, or 2 mM erythro-9-[3-(2-hydroxynonyl)]adenine (EHNA) inhibited the sliding. Thus, the pharmacological characteristics of this motility were partly similar to, although not the same as, those of the known microtubule-dependent motilities.

Changes in diadenosine tetraphosphate levels in Physarum polycephalum with different oxygen concentrations.

Cellular levels of diadenosine tetraphosphate (Ap4A) were measured, by a specific high-pressure liquid chromatography method, in microplasmodia of Physarum polycephalum subjected to different degrees of hypoxia, hyperoxia, and treatment with H2O2. Ap4A levels increased three- to sevenfold under anaerobic conditions, and the microplasmodia remained viable after such treatment. Elevated levels of Ap4A returned to the basal level within 5 to 10 min upon reoxygenation of the microplasmodia. The increases in Ap4A levels were larger in stationary-phase or starved microplasmodia than in fed, log-phase microplasmodia. The maximal increase measured in log-phase microplasmodia was twofold. No significant changes in Ap4A levels occurred in microplasmodia subjected to mild hypoxia, hyperoxia, or treatment with 1 mM H2O2. These results indicate that in P. polycephalum, Ap4A may function in the metabolic response to anaerobic conditions rather than in the response to oxidative stress.

Regulation of mitosis onset and thymidine kinase activity during the cell cycle of Physarum polycephalum plasmodia: effect of hydroxyurea.

The effects of hydroxyurea have been investigated on three events of the cell cycle, S-phase, mitosis, and the cyclic synthesis of thymidine kinase, in the synchronous plasmodium of the myxomycete Physarum. DNA synthesis was slowed down with limited action on other macromolecular syntheses and any increase of thymidine kinase that had already been triggered was indistinguishable from that of the control. When DNA synthesis was inhibited, the onset of the following cyclic increase of thymidine kinase synthesis occurred at the same time as in the control, but mitosis was delayed in a very early prophase stage. The arrest of thymidine kinase synthesis occurred after completion of the delayed mitosis. All these effects were suppressed when the action of hydroxyurea was prevented by the addition, to the medium, of the four deoxyribonucleosides. These observations show that (1). The blockage of S-phase does not prevent the nuclei from entering a very early prophase stage but does prevent them from proceeding through metaphase. (2) The transient blockage of DNA synthesis does not perturb the normal timing of the triggering of thymidine kinase synthesis. (3) The signal which triggers the arrest of thymidine kinase synthesis is postmitotic but does not require extensive DNA synthesis. The effect of hydroxyurea is not limited to an inhibition of S-phase. The blockage of DNA replication also led to the dissociation of the normal coordination between two other events of the cell cycle, mitosis and thymidine kinase synthesis. This observation could have strong implications in cell synchronization with chemical agents.

ADP-ribosylation in isolated nuclei of Physarum polycephalum.

ADP-ribosylation of histones and non-histone nuclear proteins was studied in isolated nuclei during the naturally synchronous cell cycle of Physarum polycephalum. Aside from ADP-ribosyltransferase (ADPRT) itself, histones and high mobility group-like proteins are the main acceptors for ADP-ribose. The majority of these ADP-ribose residues is NH2OH-labile. ADP-ribosylation of the nuclear proteins is periodic during the cell cycle with maximum incorporation in early to mid G2-phase. In activity gels two enzyme forms with Mr of 115,000 and 75,000 can be identified. Both enzyme forms are present at a constant ratio of 3:1 during the cell cycle. The higher molecular mass form cannot be converted in vitro to the low molecular mass form, excluding an artificial degradation during isolation of nuclei. The ADPRT forms were purified and separated by h.p.l.c. The low molecular mass form is inhibited by different ADPRT inhibitors to a stronger extent and is the main acceptor for auto-ADP-ribosylation. The high molecular mass form is only moderately auto-ADP-ribosylated.

Specific nuclear elimination in polyploid plasmodia of the slime mold Physarum polycephalum.

In growing plasmodia of the myxomycete Physarum polycephalum (G2-phase), three distinct classes of nuclei with a relative DNA content of 1x, 2x, and 4x are observed in the presumed haploid strain CL. The 2x and 4x species comprise up to 35% and 5% of the nuclei. Quantitative cytofluorometric studies of nuclei isolated in either G2- or S-phase or after FUDR treatment (G1 arrest) show that the three nuclear populations undergo a synchronous mitotic cycle and that the relative DNA content of the nuclear fractions in G-2 phase reflects the 2c, 4c, and 8c state. The heterogeneity of the nuclear population does, however, seem to be restricted to the growth phase. During a starvation period of 4 days that always preceeds sporulation (and also meiosis), the 4c nuclear population is reduced to 7%, 8c nuclei are no longer detected. These results suggest that a mechanism exists in Physarum for the selective detection and elimination of polyploid nuclei.

Effect of magnesium on the contractile behavior of actomyosin aggregates isolated from Physarum plasmodia.

The dependence on calcium concentration of the contractile behavior of actomyosin isolated from Physarum plasmodia according to Kohama & Kendrick-Jones (1986) was investigated under different magnesium conditions. The inhibitory calcium sensitivity is reduced at magnesium concentrations above or below 1 mM, i.e., contraction of actomyosin aggregates is most effectively inhibited in the presence of 1 mM calcium in combination with physiological magnesium concentrations. In the absence of calcium reactivation optimum is obtained at 8.5 mM Mg2+.

Chemically induced changes in the morphology, dynamic activity and cytoskeletal organization of Physarum cell fragments.

Spherical cell fragments derived from Physarum polycephalum by caffeine-treatment were used as an experimental system to investigate the influence of 15 externally applied substances on the general morphology, motile behavior and cytoskeletal organization of the acellular slime mold. In comparison to controls, the most obvious changes observed after chemical stimulation proved to be cytokinetic activities, ameboid-like movement phenomena, intense cell surface dynamics and formation of cytoplasmic actin fibrils. The results demonstrate the high adaptability of the microfilament system in Physarum even when subjected to extreme conditions in the external environment.

Superoxide dismutase activity and glutathione concentration during the calcium-induced differentiation of Physarum polycephalum microplasmodia.

Microplasmodia of Physarum polycephalum differentiate into spherules when the CaCl2 concentration of their nutrient medium is increased to 54mM (high-calcium). The salts starvation medium routinely used to induce differentiation contains 8mM CaCl2. This medium will not induce spherulation in the absence of a calcium salt; no other metal is essential. High-calcium also induces the spherulation of a strain of Physarum that had not been previously observed to spherulate. The striking increase in superoxide dismutase activity (SOD) and the decrease in glutathione concentration (GSH) that are characteristic of salts-induced spherulation do not occur in salts media containing high-calcium. In the absence of calcium, no significant change in SOD is observed and very little change in GSH occurs. The immediate effect of the oxidative stress associated with spherulation may be the release of calcium stores into the cytosol. The parameters modulating this stress are, in turn, sensitive to exogenous calcium concentrations.